The goals of this proposal are to determine how a histone deposition protein promotes DNA replication fork stability, and how the DNA replication machinery communicates the status of the replication fork to the replication checkpoint surveillance system, using yeast as a model system. In Aim 1, I will investigate how specific mutations in the DNA polymerase processivity clamp, PCNA, impair activation of the replication checkpoint in response to replication stress. To do this, I will determine which steps in the replication checkpoint signaling pathway are defective in these PCNA mutants. Additionally, I will perform protein and chromatin immunoprecipitation experiments to test the hypothesis that interactions between checkpoint proteins, fork-associated factors and the DNA replication fork will be perturbed in the PCNA mutants. In Aims 2 and 3, the mechanism behind replication fork collapse in cells lacking histone deposition protein Asf1 will be examined. Asfl mutant alleles specifically defective for fork stability, and a regulated Asf1 N-degron will be developed to address this question. Using these tools, fate of DNA replication forks and DNA replication proteins at stalled and moving replication forks will be examined by ChIP and 2D gel electrophoresis.